Upregulation of DR3 expression in CD4⁺ T cells promotes secretion of IL-17 in experimental autoimmune uveitis.

Purpose This study investigated the role of death receptor 3 (DR3) in experimental autoimmune uveitis (EAU). Methods EAU was induced in B10.RIII mice by subcutaneous injection of interphotoreceptor retinoid-binding protein (IRBP) 161–180 emulsified with complete Freund’s adjuvant and evaluated with clinical and histopathologic observation. Total protein of draining lymph nodes (DLNs) was extracted from the control, EAU, or recovery phase mice. CD4+ T cells were separated from lymphocytes with magnetic-assisted cell sorting. At the same time, some of the CD4+ T cells were cultured with or without recombinant TL1A (rTL1A, the DR3 ligand) for three days, and the supernatants were collected for the interleukin-17 (IL-17) test. DR3 mRNA and protein levels in CD4+ T cells and the endogenous concentration of TL1A in mice DLNs were assessed with real-time PCR or western blotting. Levels of IL-17 in the supernatants were determined with enzyme-linked immunosorbent assay. Results Histopathological and clinical data revealed severe intraocular inflammation in the immunized mice. The inflammation reached its peak on day 14 in EAU and had resolved in the recovery phase (weeks 4–5 or more after IRBP immunization). CD4+ T cells obtained from EAU (day 7 or 14) had higher levels of DR3 mRNA and protein expression compared with the control group treated with complete Freund’s adjuvant alone and the recovery group. However, the DR3 mRNA and protein levels on day 21 in EAU were similar to those observed in the control and recovery groups. The endogenous levels of TL1A were upregulated in EAU, and decreased in the recovery phase mice. Adding rTL1A increased the production of IL-17 by CD4+ T cells isolated from mice DLNs. Moreover, the increased IL-17 levels in the culture supernatant of CD4+ T cells from EAU were much higher than those from the control and recovery phase mice. However, the effects on promoting IL-17 production in TL1A-stimulated CD4+ T cells were similar between the controland recovery groups. Conclusions Our data suggest that DR3 expression is induced during EAU and may be involved in the development of this disease, possibly by promoting IL-17 secretion.

Thus far, studies have shown that CD4 + T cells are essential for the development of EAU [7,[49][50][51]. However, the regulation of DR3 expressed by CD4 + T cells or the effect of DR3 on IL-17 production has not been investigated in the EAU model. This study is the first to investigate the expression and function of DR3 in CD4 + T cells in EAU. We found that DR3 mRNA and protein levels were elevated in EAU and that stimulation with the DR3 ligand, TL1A, upregulated the secretion of IL-17 from CD4 + T cells. These results indicate that DR3 may play an important role in the development and maintenance of EAU.

METHODS
Mice and immunization: B10.RIII mice (6-8 weeks of age) were purchased from Jackson Laboratories (Bar Harbor, ME) and were housed under standard (specific pathogen-free) conditions. All animals were treated according to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. IRBP161-180 (SGIPYIISYLHPGNTILHVD) was synthesized by Shanghai Sangon Biologic Engineering Technology and Services Ltd. Complete Freund's adjuvant (CFA) containing 1.0 mg/ml Mycobacterium tuberculosis and pertussis toxin (PTX) were obtained from Sigma-Aldrich Co. (St. Louis, MO). To induce EAU, mice (8-12 weeks of age) were immunized subcutaneously with a 200 µl emulsion containing 50 µg IRBP161-180 in CFA. PTX (1.0 µg) was concurrently injected intraperitoneally as an adjuvant [52]. Control groups of mice received an emulsion of 50 µl PBS and 150 µl CFA, which was injected subcutaneously. Each experimental group consisted of 6-8 mice. Clinical examination and histopathological evaluation: After the animals were immunized, they were observed with slit lamp microscopy and ophthalmoscopy starting on day 7 until day 28. Eyes were enucleated from the control, EAU, and recovery phase mice (weeks 4-5 or more after IRBP immunization), were fixed for 1 h in 4% buffered glutaraldehyde, and were then transferred to 10% buffered formaldehyde until processing. Fixed and dehydrated tissue was embedded in paraffin and 5-to 7-µm sections were stained using a standard hematoxylin and eosin (H and E) approach. The intensity of EAU was scored from 0 to 4 in a blinded fashion according to the histopathological grading system previously described for murine EAU [50,53]. Purification of CD4+ T cells, cultivation, and medium collection: Lymphocytes were collected from mice by draining lymph nodes (DLNs, inguinal and iliac), and CD4 + T cells were isolated using a specialized kit (Miltenyi Biotec, Palo Alto, CA). Briefly, lymphocyte suspensions were incubated with CD4 MicroBeads to sort CD4 + T cells. Then T cells were incubated in Gibco RPMI 1640 medium (Invitrogen, Carlsbad, CA) with anti-CD3 (1 µg/ml) and anti-CD28 (1 µg/ml) (eBioscience, San Diego, CA), with or without recombinant TL1A (rTL1A, 100 ng/ml [29,54]; R and D Systems, Minneapolis, MN). Incubations were performed in a 24-well culture plate for 72 h at 37 °C under an atmosphere of 5% CO2. After incubation, the supernatant was collected and stored at −80 °C.

Reverse transcription PCR (RT-PCR) and real-time PCR:
Total RNA was extracted from CD4 + T cells that had been isolated from control, EAU, or recovery phase mice using the RNA extraction kit (RNeasy Mini Kit, Qiagen, Hilden, Germany). Two micrograms of total RNA from each sample were used for reverse transcription using the Superscript III Reverse Transcriptase system (Invitrogen). The following sequences of the DR3 and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) primers used for real-time PCR: 5′-GGG CTA TCC TGA TCT GTG CAT-3′ (forward primer, DR3), 5′-ATG CCA GAG GAG TTC CAA GAGT-3′ (reverse primer, DR3), 5′-GAG AAC TTT GGC ATT GTG G-3′ (forward primer, GAPDH), and 5′-ATG CAG GGA TGA TGT TCT G-3′ (reverse primer, GAPDH). The mRNA expression levels were normalized to GAPDH, which was used as a reference housekeeping gene. PCR analysis was conducted on the real-time fluorescence quantitative PCR system using SYBR Green PCR Master Mix (Qiagen) according to the manufacturer's instructions.
Western blotting analysis: Total protein of DLNs was extracted from the control, EAU, or recovery phase mice using a protein extraction kit (BioChain, Hayward, CA). CD4 + T cells were homogenized using an ultrasonicator (BANDELIN Electronic, Bberlin, Germany), and the protein lysates were prepared for western blotting (WB) analysis. Fifty micrograms of protein from each sample was subjected to sodium fodecyl sulfate-PAGE, and the separated proteins were then transferred to polyvinylidene fluoride membranes. The membranes were incubated with anti-mouse TL1A (rabbit polyclonal antibody, Abcam, Cambridge, MA), antimouse DR3 (rabbit polyclonal antibody, Santa Cruz Biotechnology, Santa Cruz, CA), or anti-β-actin (rabbit polyclonal antibody, Santa Cruz Biotechnology), followed by a secondary antibody (goat antirabbit IgG-HRP; Santa Cruz Biotechnology). Proteins were detected using the Phototope-HRP western blot detection system (Cell Signaling, Danvers, MA). Enzyme-linked immunosorbent assay: Levels of IL-17 in culture media were measured using a commercially available enzyme-linked immunosorbent assay (ELISA) kit (R and D Systems). The detection limit of the kit is 15 pg/ml. Statistical analysis: Data were expressed as mean±SD. The experimental groups were compared with Student t tests, assuming equal variances for all data. P values < 0.05 were considered to be statistically different.
Immunohistochemistry revealed severe inflammation in the posterior segment and a massive influx of inflammatory cells infiltrating the vitreous body and retina, vitritis, vasculitis, granuloma formation, and retinal photoreceptor lesions ( Figure 1B). Inflammation was initially identified on days 7-9 after immunization and reached its peak by day 14. Inflammation was then followed by a rapid resolution and recovery ( Figure 1D). As published reports have shown, there was no apparent inflammation in the control (treated with CFA only) [50,52] and recovery groups (week 4-5 or more after IRBP immunization) [53] (Figure 1A,C).

Induction of EAU increases DR3 mRNA and protein expression in CD4 + T cells:
Since previous studies suggested that CD4 + T cells play a significant role in the development and maintenance of EAU [7,49], we used CD4 microbeads to sort CD4 + T cells from mouse lymphocytes obtained by draining LNs. The mRNA and total protein were extracted from CD4 + T cells for analysis with RT-PCR and WB. We found that the DR3 mRNA and protein levels increased in CD4+ T cells from the EAU group 7 or 14 days after immunization ( Figure 2). However, there were no obvious differences in DR3 mRNA and protein levels between the controls, EAU (day 21), and recovery phase mice. We also found that the expression of DR3 in CD4 + T cells in EAU achieved the highest level at day 14, and then declined ( Figure  2). These data raise the question of the role of DR3 upregulation in EAU.

Secretion of IL-17 into CD4 + T cell culture medium is upregulated by the DR3 ligand, TL1A:
To determine the potential role for increased DR3 expression in EAU, we chose the cells of the most severe inflammatory mice (day 14) to represent the EAU [53] as shown in Figure 1 and Figure 2. At first, we detected the endogenous levels of TL1A in mice DLNs with WB and found that the concentration of TL1A in EAU was upregulated; then it decreased in the recovery phase mice (Figure 3). After that, we added recombinant TL1A into the medium of the CD4 + T cells. Since under conditions of suboptimal anti-CD3/CD28 stimulation CD4 + T cells are thoroughly activated [35], we included anti-CD3/CD28 in the medium. We then measured IL-17 in the culture supernatants with ELISA and found that TL1A interaction with DR3 clearly promoted IL-17 production by CD4 + T cells compared with the media without TL1A in the control, EAU, or recovery mice ( Figure 4A). This effect was significantly more pronounced in CD4 + T cells obtained from the DLNs of the EAU mice. Then we calculated the increased concentration of IL-17 after stimulated by TL1A for 2 h, and found that the elevated IL-17 levels in the culture supernatant in the EAU group were much higher, about fourfold, than that in the other two groups ( Figure 4B). However, the increased levels of IL-17 stimulated by TL1A in the control group were similar to those in the recovery group ( Figure 4B).

DISCUSSION
Uveitis is considered a typical T-cell mediating, organspecific, autoimmune disease. EAU in B10.RIII mice induced by IRBP161-180, the best-studied model of uveitis recently shown to be IL-17 driven [23,44,55], has commonly been described as a monophasic disease [56], with a clinical peak about 2 weeks after immunization [37,57,58], then followed by remission and tolerance to reinduction; namely, EAU is a self-limited disease [1]. Similar to EAU, human posterior uveitis is also characterized by a bilateral granuloma, vasculitis, retinal lesions, and so on; however, human posterior uveitis often follows a relapsing and remitting course [59][60][61], of which the etiology and pathology are still elusive [62].
To further investigate its function, we analyzed the role of DR3 in EAU. First, we examined the expression of DR3 in mice. The DR3 mRNA and protein levels in the CD4 + T cells of EAU mice were upregulated compared with the recovery phase mice and controls. Since DR3 expression coincides with the rapid induction of its ligand (TL1A) expression [29,32] and DR3 binding to TL1A regulates T-cell activation and expansion [27,63], we examined whether TL1A was also elevated in EAU. Results showed that the endogenous TL1A levels were significantly increased in EAU compared with the recovery phase mice and controls. These data imply that DR3 needs to be coupled with its ligand TL1A to execute its function.
Several studies show that Th17 cells or IL-17 are associated with ocular inflammatory diseases such as uveitis [60,64,65] and CD4 + T cells are capable of producing IL-17 [12]. Therefore, we studied the effect of DR3 on IL-17 production in CD4+ T cells and try to explain the mechanism of DR3 function in EAU. We found that DR3 interaction with TL1A could induce the increase of IL-17 production by CD4 + T cells. This is consistent with other findings that DR3-TL1A interaction regulates the Th17 cell function and IL-17mediated autoimmune disease [35]. In summary, our study showed that increased DR3 production may be associated with the development of EAU in mice. These results indicate that stimulation of DR3 with TL1A could increase IL-17 production, with the suggestion that the DR3:TL1A signaling pathway may be involved in the pathogenesis of autoimmune uveitis.
In future research, we plan to determine how the interaction of TL1A with DR3 can increase the secretion of IL-17. Our studies will focus on elucidating cell signaling pathways that are activated in response to TL1A binding to DR3 and on the possibility of the production of cytokines other than IL-17. In addition, we want to knock out the DR3 gene in B10.RIII mice to observe whether the EAU model can still be induced. These studies will provide valuable new insights, and ultimately, we hope that elucidation of these mechanisms will enable the development of new therapeutic methods to treat human autoimmune and inflammatory diseases.